CN116199742B - A method for preparing recombinant human chorionic gonadotropin Fc fusion protein - Google Patents
A method for preparing recombinant human chorionic gonadotropin Fc fusion protein Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract 5
- 108010062540 Chorionic Gonadotropin Proteins 0.000 title abstract 2
- 102000011022 Chorionic Gonadotropin Human genes 0.000 title abstract 2
- 108091006020 Fc-tagged proteins Proteins 0.000 title abstract 2
- 229940084986 human chorionic gonadotropin Drugs 0.000 title abstract 2
- 108020001507 fusion proteins Proteins 0.000 claims abstract 19
- 102000037865 fusion proteins Human genes 0.000 claims abstract 19
- 108010076504 Protein Sorting Signals Proteins 0.000 claims abstract 8
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- 230000001737 promoting effect Effects 0.000 claims abstract 3
- 230000028327 secretion Effects 0.000 claims abstract 3
- 108090000623 proteins and genes Proteins 0.000 claims 9
- 239000002773 nucleotide Substances 0.000 claims 2
- 125000003729 nucleotide group Chemical group 0.000 claims 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 230000003248 secreting effect Effects 0.000 abstract 2
- 108060001084 Luciferase Proteins 0.000 abstract 1
- 239000005089 Luciferase Substances 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 abstract 1
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Abstract
本发明公开了一种重组人绒毛膜促性腺激素Fc融合蛋白制备方法。本发明提供了SEQ ID No.5所示信号肽或其相关生物材料在如下任一中的应用：A1)制备HCG蛋白或其融合蛋白；A2)提高HCG蛋白或其融合蛋白的表达量或产量；A3)促进HCG蛋白或其融合蛋白分泌到宿主细胞外；本发明采用HCG‑Fc融合蛋白的三种信号肽用于引导HCG‑Fc融合蛋白真核细胞分泌表达，人工改造信号肽SP1分泌表达量显著优于抗体轻链信号肽SP2和荧光素酶信号肽SP3，更适用于大规模工业化生产，降低生产成本。
The present invention discloses a method for preparing a recombinant human chorionic gonadotropin Fc fusion protein. The present invention provides the use of a signal peptide shown in SEQ ID No.5 or its related biological materials in any of the following: A1) preparing HCG protein or its fusion protein; A2) increasing the expression level or yield of HCG protein or its fusion protein; A3) promoting the secretion of HCG protein or its fusion protein outside the host cell; the present invention uses three signal peptides of HCG-Fc fusion protein to guide the secretory expression of HCG-Fc fusion protein eukaryotic cells, and the secretory expression of the artificially modified signal peptide SP1 is significantly better than that of the antibody light chain signal peptide SP2 and the luciferase signal peptide SP3, which is more suitable for large-scale industrial production and reduces production costs.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method of recombinant human chorionic gonadotrophin Fc fusion protein.
Background
Human Chorionic Gonadotropin (HCG) is a glycoprotein hormone synthesized by syntrophic cells and consists of two different alpha and beta subunits, wherein the alpha subunit is basically similar to follicle stimulating Hormone (HCG), luteinizing Hormone (LH), thyroid Stimulating Hormone (TSH) and the like secreted by a pituitary, so that cross reactions can occur between the alpha subunit and the beta subunit, and the structures of the beta subunit are different.
HCG has the functions of 1) HCG and LH, maintaining the life span of menstrual corpus luteum, increasing the menstrual corpus luteum to the gestational corpus luteum, 2) promoting the aromatic conversion of androgens to estrogens and stimulating the formation of progesterone, 3) inhibiting the stimulation of phytolectin on lymphocytes, adsorbing human chorionic gonadotropin on the surface of trophoblasts to prevent embryonic trophoblasts from being attacked by maternal lymphocytes, 4) LH-like function, and stimulating the testicle of a fetus to secrete testosterone before the fetal pituitary secretes LH to promote male sexual differentiation, 5) promoting gonadal development, stimulating the activity of testicular intermediate cells for males, and increasing the secretion of male hormone (testosterone). Clinically, the HCG product is suitable for infertility, luteal phase deficiency, functional uterine bleeding, threatened abortion or habitual abortion and the like.
Disclosure of Invention
It is an object of the present invention to provide the use of SP1 signal peptide or biological material related thereto.
The invention provides the use of an SP1 signal peptide or a biomaterial related thereto in any one of the following A1) -A3):
a1 Preparing HCG protein or fusion protein thereof;
a2 Increasing the expression level or yield of HCG protein or a fusion protein thereof;
a3 Promoting secretion of HCG protein or fusion protein thereof to the outside of the host cell;
the amino acid sequence of the SP1 signal peptide is shown as SEQ ID No. 5.
The above HCG fusion protein has an a chain which is an α chain of HCG and a B chain which is a β chain of HCG linked to a tag. The above-mentioned tag may be any one or any combination of Fc, HSA, CTP or XTEN, preferably the tag is an Fc tag.
The β chain of HCG linked to the Fc tag is a C-terminal linked tag of the β chain of HCG.
In the above application, the biological material related to the SP1 signal peptide is the coding gene of the SP1 signal peptide or an expression cassette, a recombinant vector, a recombinant bacterium or a recombinant cell line containing the coding gene.
In the application, the amino acid sequence of the A chain in the HCG fusion protein is shown as SEQ ID No. 1;
the amino acid sequence of the B chain in the HCG fusion protein is shown as SEQ ID No. 2.
It is another object of the present invention to provide HCG fusion proteins.
The HCG fusion protein provided by the invention is B1) or B2) as follows:
B1 The A chain of the HCG fusion protein is an alpha chain of HCG, and the B chain is a beta chain of HCG connected with a label;
B2 The A chain of the HCG fusion protein is an alpha chain of HCG connected with the SP1 signal peptide, and the B chain is a beta chain of HCG connected with the SP1 signal peptide and the tag;
the amino acid sequence of the SP1 signal peptide is shown as SEQ ID No. 5.
The above-mentioned tag may be any one or any combination of Fc, HSA, CTP or XTEN, preferably the tag is an Fc tag.
Preferably, the HCG fusion protein shown in B1) is an HCG-Fc fusion protein, HCG is human HCG, specifically, an Fc tag is fused to the carbon end of a beta chain of a human HCG molecule to form a B chain of HCG-Fc, the B chain forms a homodimer through disulfide bonds of an Fc structure, and an alpha chain of the HCG molecule (namely, an A chain of the HCG-Fc) is combined with the B chain through a non-covalent bond to form a bivalent HCG molecule.
Preferably, the HCG fusion protein shown in B2) is SP1-HCG-Fc fusion protein, HCG is human HCG, specifically, fc tag is fused to the carbon end of beta chain of human HCG molecule, SP1 signal peptide is connected to the nitrogen end of beta chain of human HCG molecule to form B chain of SP1-HCG-Fc, B chain forms homodimer through disulfide bond of Fc structure, SP1 signal peptide is connected to the nitrogen end of alpha chain of human HCG molecule to form A chain of SP1-HCG-Fc, and A chain of SP1-HCG-Fc is combined with B chain through non-covalent bond to form bivalent HCG molecule.
Further preferably, the above HCG-Fc fusion protein, wherein the Fc is derived from IgG1-Fc and the HCG is human HCG. Wherein Fc is IgG1-Fc, specifically, CH2 and CH3 regions of human IgG1-Fc are adopted, and Lys (K) amino acid at the carbon terminal is removed, so that the charge heterogeneity of molecules is reduced.
The HCG fusion protein is formed by non-covalent combination of an alpha chain of HCG and a beta chain of HCG connected with an Fc tag;
the above HCG fusion protein is formed by non-covalent bonding of an α chain of HCG linked to the SP1 signal peptide and a β chain of HCG linked to the SP1 signal peptide and the Fc tag.
The Fc tag is at the C-terminus of the α chain of HCG;
the SP1 signal peptide is linked to the N-terminus of the alpha or beta chain of HCG.
In the above-mentioned HCG fusion protein,
B1 The amino acid sequence of the A chain of the HCG fusion protein is shown as SEQ ID No.1, and the amino acid sequence of the B chain of the HCG fusion protein is shown as SEQ ID No. 2;
Or B2), the amino acid sequence of the A chain of the HCG fusion protein is shown as SEQ ID No.7, and the amino acid sequence of the B chain of the HCG fusion protein is shown as SEQ ID No. 8.
Biological materials associated with the fusion proteins described above are also within the scope of the present invention.
The biological material is the encoding gene of the fusion protein or an expression cassette, a recombinant vector, a recombinant bacterium or a recombinant cell line containing the encoding gene.
The encoding gene of the fusion protein is any one of the following C1-C4:
C1:B1) the nucleotide sequence of the encoding gene of the A chain of the HCG fusion protein is shown as SEQ ID No.3, and the nucleotide sequence of the encoding gene of the B chain of the HCG fusion protein is shown as SEQ ID No. 4;
The nucleotide sequence of the encoding gene of the A chain of the HCG fusion protein is shown as SEQ ID No.9, and the nucleotide sequence of the encoding gene of the B chain of the HCG fusion protein is shown as SEQ ID No. 10.
C3 a DNA molecule which hybridizes under stringent conditions to a DNA molecule defined in any one of C1 or C2 and which encodes the above-described fusion protein;
c4 is a DNA molecule which has more than 75% identity with a DNA sequence defined by a DNA molecule defined by either C1 or C2 and encodes the fusion protein.
In the above nucleic acid molecules or coding genes, identity refers to the identity of nucleotide sequences. The identity of nucleotide sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, setting Expect to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per residue gap cost and Lambda ratio to 11,1 and 0.85, respectively (default values), the identity of a pair of nucleotide sequences is searched for, and calculation is performed, and then the value (%) of the identity can be obtained.
The above nucleic acid molecule or encoding gene may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identical to the above 90% or more.
In a specific embodiment of the present invention, the recombinant expression plasmid pCGS-SP 1-HCG-Fc is a recombinant expression plasmid obtained by inserting the DNA fragment shown in SEQ ID No.9 into the multiple cloning site (e.g., hindIII and XhoI) of pCGS vector, and inserting the DNA fragment shown in SEQ ID No.10 into the multiple cloning site (e.g., bstBI and PacI) of pCGS vector.
Wherein the expression cassette refers to a DNA capable of expressing the fusion protein of the first aspect in a host cell, which DNA comprises not only a promoter for initiating transcription of the target gene, but also a terminator for terminating transcription of the target gene. Further, the expression cassette may also include an enhancer sequence.
Wherein the host cell is a eukaryotic host cell.
Further, the eukaryotic host cell may be HEK293 cells, CHO cells, yeast cells, insect cells, etc., and in particular embodiments of the invention, the host cell is an Expi293F cell.
The use of the above-mentioned biomaterials in any of the following A1) -A3) is also within the scope of the present invention:
a1 Preparing HCG protein or fusion protein thereof;
a2 Increasing the expression level or yield of HCG protein or a fusion protein thereof;
A3 Promoting secretion of HCG proteins or fusion proteins thereof out of the host cell.
It is still another object of the present invention to provide a method for preparing HCG protein.
The method provided by the invention comprises the following steps of expressing the encoding gene of the fusion protein in host bacteria or host cells to obtain HCG protein.
The method comprises the following steps:
1) Introducing encoding genes of A chain and B chain in the fusion protein into host bacteria to obtain recombinant bacteria;
2) Culturing the recombinant strain to obtain HCG protein.
The encoding genes of the A chain and the B chain in the fusion protein are introduced into host bacteria through recombinant plasmid pCGS-SP 1-HCG-Fc;
In a specific embodiment of the invention, the host cell is specifically an Expi293F cell.
In the step 2), the culture is terminated when the cell viability is reduced to 65% -75%.
After step 2), step 3) is also included, wherein HCG-Fc fusion Protein is obtained from the supernatant of the cultured product after the culture, the culture is collected and centrifuged at 3500g for 30min, and the supernatant is collected for ultrafiltration concentration, protein A4 FF filler affinity chromatography and ultrafiltration displacement.
The invention designs an HCG-Fc fusion protein which is used as a candidate molecule of long-acting HCG and carries out signal peptide optimization experiments on the basis. The artificial engineering signal peptide SP1 (SEQ ID No. 5) is found to guide the secretory expression of eukaryotic cells of the HCG-Fc fusion protein. However, the secretion of the SP1 signal peptide was not enhanced for all proteins (experience with the different protein expression accumulated in this laboratory indicates that the expression of the CD19 protein, as guided by the SP1 signal peptide, is very low), which was also relevant for the experiments of the present invention. Therefore, the artificial transformation signal peptide SP1 is obviously superior to the antibody light chain signal peptide SP2 and the luciferase signal peptide SP3 in guiding the eukaryotic cell secretion expression quantity of the HCG-Fc fusion protein, is more suitable for large-scale industrial production, and reduces the production cost.
Drawings
FIG. 1 is a diagram showing the prediction of the secretory expression of an artificially engineered signal peptide SP 1. SP (Sec/SPI) is the predicted signal peptide probability, CS is the predicted signal peptidase cleavage site, and OTHERS represents the probability of non-signal peptide. The ordinate indicates the probability that the amino acid sequence is the predicted structure, and the abscissa indicates the protein amino acid sequence. Wherein A is a HCG-Fc fusion protein A chain prediction graph, and B is a HCG-Fc fusion protein B chain prediction graph.
FIG. 2 is a diagram showing the identification of the construction of the recombinant expression plasmid of HCG-Fc fusion protein. Wherein the A chain is cut by HindIII and XhoI, the B chain is cut by BstBI and PacI, 1 is pCGS-SP 1-HCG-Fc (artificial engineered signal peptide SP1 expression plasmid), 2 is pCGS-SP 2-HCG-Fc (antibody light chain signal peptide SP2 expression plasmid), and 3 is pCGS-SP 3-HCG-Fc (luciferase signal peptide SP3 expression plasmid).
FIG. 3 is a diagram showing non-reducing SDS-PAGE identification of cell supernatants of HCG-Fc fusion proteins. Wherein 1 is pCGS-SP 1-HCG-Fc/293,2 is pCGS-SP 2-HCG-Fc/293,3 is pCGS-SP 3-HCG-Fc/293,4, and is a negative control group without plasmid transfection.
FIG. 4 is a graph showing the analysis of ash removal on cell secretion of HCG-Fc fusion protein. Wherein, the arrow indicates the peak of HCG-Fc fusion protein (coinciding with the negative background protein band), A is pCGS-SP 1-HCG-Fc/293, B is pCGS-SP 2-HCG-Fc/293, C is pCGS-SP 3-HCG-Fc/293, D is a negative control group without plasmid transfection.
FIG. 5 shows SDS-PAGE purification identification of HCG-Fc fusion protein. Wherein NR is non-reducing SDS-PAGE, R is reducing SDS-PAGE,1 is SP1-HCG-Fc,2 is SP2-HCG-Fc, and 3 is SP3-HCG-Fc.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The main reagents and their manufacturer information in the following examples are as follows:
pCGS3 expression vector Merck company;
HindIII NEB Co;
XhoI NEB Co;
BstBI NEB Co;
PacI NEB company;
GXL Premix, TAKARA;
DNA Ligation Kit Ver.2.1, TAKARA Co;
Expi293F TM Cells, thermo Fisher Co;
Expi293 TM Expression Medium ThermoFisher company;
ExpiFectamine TM 293 Transfection Kit:Thermo Fisher company;
Opti-MEM TM I Reduced Serum Medium, thermo Fisher Co;
PageRuler TM pre-dyeing protein molecular weight standard, 10 to 180kDa:Thermo Fisher company;
protein A4 FF packing purification kit is manufactured and bioengineered (Shanghai) Co., ltd;
amicon Ultra-15 centrifugal filtration device Millipore company;
amicon Ultra-0.5 centrifugal filtration device: millipore company;
PBS pH7.4 (1X) Gibco company;
a gel imaging system, protein Simple company;
cell counter, roche company;
Ultra clean bench, available from air technologies, inc. of Antai, suzhou;
An electric heating thermostatic water bath pot FISHER SCIENTIFIC company;
CO 2 constant temperature shaker CRYSTAL;
ext> theext> HYGext> -ext> Aext> fullext> -ext> constantext> temperatureext> shakingext> flaskext> cabinetext> isext> aext> Taicangext> cityext> experimentalext> facilityext> factoryext>;ext>
DYY-6C electrophoresis apparatus, six instrument factories in Beijing;
DYCP-31DN horizontal electrophoresis tank, six instrument factories in Beijing;
Micropipettes Eppendorf Corp.
Example 1 Signal peptide prediction
The efficiency of artificially modified signal peptide SP1 is predicted by adopting SignalP-5.0 online software, namely, the A chain amino acid sequence (SEQ ID No. 7) and the B chain amino acid sequence (SEQ ID No. 8) of the SP1-HCG-Fc fusion protein are respectively input into websites https:// services. As shown in FIG. 1, the structural probabilities of SP1 signal peptide of the A chain and the B chain of HCG-Fc are 99.95 and 99.80%, the cleavage probabilities of signal peptide are 94.97% and 85.53%, and the cleavage sites are located between 19 th amino acid and 20 th amino acid, which is consistent with expectations.
In conclusion, the signal peptide SP1 has high cleavage efficiency, and is beneficial to the secretion and expression of HCG-Fc fusion protein (figure 1).
The amino acid sequence of the signal peptide SP1 is SEQ ID No.5, and the nucleotide sequence of the encoding gene is SEQ ID No.6.
Example 2 construction of recombinant expression plasmid
1. SP1-HCG-Fc Gene Synthesis
The entrusted Bailey organisms optimize codons of the A chain (SEQ ID No. 7) and the B chain (SEQ ID No. 8) of the SP1-HCG-Fc (SP 1 signal peptide fusion HCG-Fc) fusion protein, the optimized nucleotide sequences of the A chain and the B chain are SEQ ID No.9 and SEQ ID No.10 respectively, and the delivery cloning plasmids are named pUC57-SP1-HCGA and pUC57-SP1-HCGB-Fc respectively.
2. SP2-HCG-Fc fragment amplification
The A-chain nucleotide sequence (SP 2-HCGA, SEQ ID No. 11) and the B-chain nucleotide sequence (SEQ ID No. 12) of SP2-HCGB-Fc were amplified using the above-described synthesized pUC57-SP1-HCGA and pUC57-SP1-HCGB-Fc as templates, respectively. The method comprises the following steps:
pUC57-SP1-HCGA is used as a template, a first round of amplification is carried out on the primer 1 and the primer 5 to obtain a fragment 1, a second round of amplification is carried out on the primer 3 and the primer 5 to obtain a fragment 2, and the fragment 2 is the SP2-HCGA target fragment (marked as an A chain of SP 2-HCG-Fc).
PUC57-SP1-HCGB-Fc is used as a template, a first round of amplification is carried out on the primer 2 and the primer 6 to obtain a fragment 3, a second round of amplification is carried out on the primer 4 and the primer 6 to obtain a fragment 4, and the fragment 4 is the SP2-HCGB-Fc target fragment (marked as a B chain of SP 2-HCG-Fc).
The primer sequences used for the PCR amplification were as follows:
primer 1:5'-CTGGGCCTGCTGCTGCTGTGGCTGACCGACGCCAGATGCGCCCCCGACGTGCAGGACTG-3';
Primer 2:5'-CTGGGCCTGCTGCTGCTGTGGCTGACCGACGCCAGATGCAGCAAGGAGCCCCTGAG-3';
Primer 3:5'-CCCAAGCTTGCCGCCACCATGAGCGTGCCAACCCAGGTGCTGGGCCTGCTGCTGCT-3';
Primer 4:5'-ACGAAGTTCGAAGCCGCCACCATGAGCGTGCCAACCCAGGTGCTGGGCCTGCTGCTGCT-3';
primer 5:5'-CCCGCTCGAGTTAGCTCTTGTGGTAGTAGCAGGTG-3';
Primer 6:5'-CCTTAATTAATTAGCCGGGGCTCAGG-3'.
3. SP3-HCG-Fc fragment amplification
The A-strand nucleotide sequence (SP 3-HCGA, SEQ ID No. 13) and the B-strand nucleotide sequence (SP 3-HCGB-Fc, SEQ ID No. 14) of SP3-HCG-Fc were amplified using the above-described two synthesized pUC57-SP1-HCGA and pUC57-SP1-HCGB-Fc as templates, respectively. The method comprises the following steps:
pUC57-SP1-HCGA is used as a template, a first round of amplification is carried out on the primer 5 and the primer 7 to obtain a fragment 5, a second round of amplification is carried out on the primer 5 and the primer 9 to obtain a fragment 6, and the fragment 6 is the SP3-HCGA target fragment (marked as an A chain of SP 3-HCG-Fc).
PUC57-SP1-HCGB-Fc is used as a template, a first round of amplification is carried out on the primer 6 and the primer 8 to obtain a fragment 7, a second round of amplification is carried out on the primer 6 and the primer 10 to obtain a fragment 8, and the fragment 8 is the SP3-HCGB-Fc target fragment (marked as a B chain of SP 3-HCG-Fc).
The primer sequences used for the PCR amplification were as follows:
primer 5:5'-CCCGCTCGAGTTAGCTCTTGTGGTAGTAGCAGGTG-3';
primer 6:5'-CCTTAATTAATTAGCCGGGGCTCAGG-3';
primers 7:5'-GTTTGCTCTGATTTGTATTGCCGTGGCTGAGGCCGCCCCCGACGTGCAGGACTG-3';
Primer 8:5'-GGTGTTGTTTGCTCTGATTTGTATTGCCGTGGCTGAGGCCAGCAAGGAGCCCCTGAGG-3';
Primer 9:5'-CCCAAGCTTGCCGCCACCATGGGGGTGAAGGTGTTGTTTGCTCTGATTTGTATTGC-3';
Primer 10:5'-ACGAAGTTCGAAGCCGCCACCATGGGGGTGAAGGTGTTGTTTGCTCTGATTTGTATTGC-3'.
4. Construction of HCG-Fc fusion protein recombinant expression plasmid
The A chain of HCG-Fc fused with different connecting peptides amplified by the above-mentioned first, second and third genes, SP1-HCG-Fc, SP2-HCG-Fc, SP3-HCG-Fc and pCGS were cloned into pCGS expression vector named pCGS3-SP1-HCGA, pCGS3-SP2-HCGA, pCGS3-SP3-HCGA intermediate vector by HindIII and XhoI double cleavage, respectively.
The B chain of HCG-Fc fused with different connecting peptides amplified by the above-mentioned first, second and third genes, namely the B chain of SP1-HCG-Fc, the B chain of SP2-HCG-Fc, the B chain of SP3-HCG-Fc, pCGS-SP 1-HCGA, pCGS3-SP2-HCGA and pCGS3-SP3-HCGA intermediate vectors constructed above were subjected to double cleavage by BstBI and PacI, and a DNA ligation kit (TAKARA) was cloned into three intermediate vectors named pCGS-SP 1-HCG1-Fc, pCGS-SP 2-HCG1-Fc and pCGS-SP 3-HCG1-Fc recombinant expression plasmids, respectively.
The results of the digestion identification of the three recombinant expression plasmids are shown in FIG. 2, and the results show that 1 is pCGS-SP 1-HCG-Fc,2 is pCGS-SP 2-HCG-Fc,3 is pCGS-SP 3-HCG-Fc, and the digestion band size meets the expectations.
The recombinant expression plasmid pCGS-SP 1-HCG-Fc has the structure described by inserting the DNA fragment shown in SEQ ID No.9 between the HindIII and XhoI cleavage sites of pCGS vector, wherein the 1 st to 6 th sites of SEQ ID No.9 are HindIII cleavage sites, 7 to 15 are Kozak sequences, 16 to 72 are the coding genes of the natural SP1 signal peptide, 73 to 348 are the A chain coding genes of HCG-Fc (only the alpha chain coding genes of HCG), 349 to 351 are stop codons, 352 to 357 are XhoI cleavage sites, the 1 st to 6 th sites of SEQ ID No.10 are BstBI cleavage sites, 7 to 15 are Kozak sequences, 16 to 72 are the coding genes of the natural SP1 signal peptide, 73 to 1185 th sites are the B chain coding genes of HCG-Fc (73 to 507 are the beta chain of HCG, 508 to 1185 are the beta chain coding genes of HCG), and the DNA fragment shown in SEQ ID No.10 is inserted between the BstBI and PacI cleavage sites of pCGS vector, wherein the 1 to 6 th sites of SEQ ID No.10 are BstBI cleavage sites, 7 to 15 are Kozak sequences, 16 to 72 are the beta chain coding genes of HCG-Fc.
SEQ ID No.9 encodes the A chain of the SP1-HCG-Fc protein shown in SEQ ID No. 7. The SP1 signal peptide is arranged at the 1 st-19 th positions of SEQ ID No.7, the A chain of HCG-Fc is arranged at the 20 th-111 th positions, and the B chain of protein SP1-HCG-Fc shown in SEQ ID No.8 is encoded by SEQ ID No. 10. The SP1 signal peptide is shown in SEQ ID No.8 at positions 1-19, the B chain of HCG-Fc at positions 20-390 (the beta chain of HCG is shown in 20-164, and the Fc structure is shown in 165-390).
The recombinant expression plasmid pCGS-SP 2-HCG-Fc has the structure described by inserting the DNA fragment shown in SEQ ID No.11 between the HindIII and XhoI cleavage sites of pCGS vector, wherein the 1 st to 6 th sites of SEQ ID No.11 are HindIII cleavage sites, the 7 th to 15 th sites are Kozak sequences, the 16 th to 75 th sites are the coding genes of the natural SP2 signal peptide, the 76 th to 351 th sites are the A chain coding genes of HCG-Fc (only the alpha chain coding genes of HCG), the 352 th to 354 th sites are stop codons, 355-360 are XhoI cleavage sites, and the DNA fragment shown in SEQ ID No.12 is inserted between the BstBI and PacI cleavage sites of pCGS vector, wherein the 1 st to 6 th sites of SEQ ID No.12 are BstBI cleavage sites, the 7 to 15 th sites are Kozak sequences, the 16 to 75 th sites are the coding genes of the natural SP2 signal peptide, the 76 th to 1188 th sites are the B chain coding genes of HCG-Fc (76 to 510 th beta chain coding genes of HCG, the 511 to 1188 th sites are the beta chain coding sites of PacI), and the PacI is the PacI cleavage site of the PacI of 1199.
The recombinant expression plasmid pCGS-SP 3-HCG-Fc has the structure described by inserting the DNA fragment shown in SEQ ID No.13 between the HindIII and XhoI cleavage sites of pCGS vector, wherein the 1 st to 6 th sites of SEQ ID No.13 are HindIII cleavage sites, the 7 th to 15 th sites are Kozak sequences, the 16 th to 66 th sites are the coding genes of the natural SP3 signal peptide, the 67 th to 342 th sites are the A chain coding genes of HCG-Fc (only the alpha chain coding gene of HCG), the 343 th to 345 th sites are stop codons, the 346 th to 351 th sites are XhoI cleavage sites, the 1 st to 6 th sites of SEQ ID No.14 are BstBI cleavage sites, the 7 th to 15 th sites are Kozak sequences, the 16 th to 66 th sites are the coding genes of the natural SP3 signal peptide, the 67 th to 1179 th sites are the B chain coding genes of HCG-Fc (67 th to 501 th beta chain of HCG, 502 th to 1189 th sites are the beta chain coding genes of HCG), and the DNA fragment shown in SEQ ID No.14 is inserted between the BstBI and PacI cleavage sites of pCGS vector, and PacI is cut into the structure 0-1180.
The recombinant expression plasmids pCGS-SP 1-HCG-Fc, pCGS3-SP2-HCG-Fc and pCGS-SP 3-HCG-Fc express fusion proteins SP1-HCG-Fc, SP2-HCG-Fc and SP3-HCG-Fc, respectively, and these fusion proteins are secreted outside cells and are fusion proteins HCG-Fc.
The amino acid sequence of the A chain of the fusion protein HCG-Fc is SEQ ID No.1, the amino acid sequence of the B chain of the HCG-Fc is SEQ ID No.2, the nucleotide sequence of the encoding gene of the A chain of the HCG-Fc is SEQ ID No.3, and the nucleotide sequence of the encoding gene of the B chain of the HCG-Fc is SEQ ID No.4.
Example 3 HCG-Fc fusion protein expression
1. Host cells
Seed stock cells of host cells, expi293F, were taken from a liquid nitrogen tank, thawed rapidly in a 37 ℃ water bath, and the thawed cell suspension was aseptically transferred to a 125ml vial containing 30ml of pre-warmed complete growth medium under shaking conditions of 37 ℃,8% CO 2, 120rpm, amplitude 25mm, humidity > 80%. And taking the cell suspension after 15-30min to detect the cell density and the activity.
When the cell viability was recovered by 90% or more, the cell density was 5X 10 6 cells/ml, and the cells were inoculated and amplified at 0.5X 10 6 cells/ml.
2. Cell transfection
Host cells were transfected with pCGS-SP 1-HCG-Fc, pCGS-SP 2-HCG-Fc and pCGS-SP 3-HCG-Fc constructed in example 2, respectively.
1. The day before transfection
Cells were re-seeded at 3X 10 6 cells/ml 24h before transfection and cultured for 24h.
2. Day of transfection
(1) The cell density should reach 5.5X10 6 cells/ml, and the activity rate should be more than or equal to 95%. The cells were diluted to 3X 10 6 cells/ml with fresh pre-warmed complete growth medium.
(2) Preparation of transfection reagent and DNA Complex
1) DNA dilution
The plasmids (pCGS-SP 1-HCG-Fc, pCGS-SP 2-HCG-Fc or pCGS-SP 3-HCG-Fc constructed in example 2) were diluted to 1. Mu.g/. Mu.l with sterile water, and the amount of plasmid required for transfection of 50ml of cells was taken up in an amount of 1ml of cells to 1. Mu.g of plasmid, i.e.50. Mu.l of plasmid was added to 3ml of Opti-MEM TM I Reduced Serum medium for later use.
2) Transfection reagent dilution
Before use, the transfection Reagent ExpiFectamine and TM Reagent are mixed gently upside down, and the amount of the transfection Reagent required for transfecting 50ml of cells, namely 160 mu l ExpiFectamine293 TM Reagent, is mixed gently upside down in 2.8ml Opti-MEMTM I Reduced Serum medium, and the mixture is left standing for 5min at room temperature.
3) Adding the diluted transfection reagent into the plasmid, gently mixing the mixture upside down, and reacting the mixture at room temperature for 20min. The mixed transfection reagent and DNA complex is slowly added to the cell culture. Culturing at 37 deg.C, 8% CO 2, 120rpm, amplitude 25mm, humidity not less than 80%.
3. First day after transfection
22H after transfection, enhancers were added in the amount of 50ml cells transfected. That is, 300. Mu l ExpiFectamine TM 293Transfection Enhancer 1 and 3ml ExpiFectamine TM 293Transfection Enhancer 2 were mixed uniformly and then slowly added to the cell culture.
4. Culture supernatant collection
Cell viability was monitored daily after transfection and the culture was terminated for 4 days when viability was reduced to 65% -75%. Cultures were collected by centrifugation at 3500g for 30min and supernatants were collected under the names pCGS-SP 1-HCG-Fc/293 (containing the HCG-Fc fusion protein expressed by secretion of the signal peptide SP 1), pCGS-SP 2-HCG-Fc/293 (containing the HCG-Fc fusion protein expressed by secretion of the signal peptide SP 2) and pCGS-SP 3-HCG-Fc/293 (containing the HCG-Fc fusion protein expressed by secretion of the luciferase signal peptide SP 3), respectively.
SDS-PAGE identification and gray scale analysis were performed on the supernatant.
SDS protein electrophoretogram greyscale analysis Image J software was used. The method comprises the steps of converting Image, type, 32-Bit into a gray level map, processing, subtract Background, OK and removing background color, selecting lanes by a rectangular tool, determining analysis lanes by analysis, gel, SELECT FIRST LANE, repeatedly selecting a plurality of lanes for simultaneous analysis, generating peak areas by analysis, gel and Plot Lane, selecting a peak map corresponding to a target strip by a linear tool, and calculating the peak map areas by a Wand tool to obtain the total protein proportion of target protein expression.
As a result, as shown in FIGS. 3 and 4, it was found that the expression level of the HCG-Fc fusion protein in which the signal peptide SP1 was expressed by secretion was increased 2.06-fold and 1.22-fold over the light chain signal peptide SP2 and the luciferase signal peptide SP3, respectively.
In summary, it was found that the artificial signal peptide SP1 of the present invention has a higher secretory expression amount yield of the HCG-Fc fusion protein than the light chain signal peptide SP2 and the luciferase signal peptide SP 3.
Example 4 protein purification
1. Ultrafiltration concentration
The supernatants from samples (i.e., the supernatant obtained by "collecting cultures after centrifugation at 3500g for 30min" in step 4 of example 3) were pCGS-SP 1-HCG-Fc/293 (containing the HCG-Fc fusion protein expressed by secretion of the signal peptide SP 1), pCGS-SP 2-HCG-Fc/293 (containing the HCG-Fc fusion protein expressed by secretion of the signal peptide SP 2) and pCGS-SP 3-HCG-Fc/293 (containing the HCG-Fc fusion protein expressed by secretion of the luciferase signal peptide SP 3), and concentrated by centrifugation at 6000g for 20min using an Amicon Ultra-15 centrifugation filter (Millipore Co.) and the final cell supernatants were concentrated to 20 to 30ml.
2. Affinity chromatography
The ultrafiltration concentrated supernatant obtained in the first step is purified by using a Protein A4 FF packing purification kit (manufacturing engineering). The supernatant and Binding/Wash Buffer are mixed uniformly according to the volume ratio of 1:1, and the mixture is kept stand for 20min for full incubation. Double column volume Binding/Wash Buffer equilibrates the column and Buffer flows through the pre-packed column by gravity flow. Adding the ultrafiltration concentrated supernatant and Binding/Wash Buffer mixed solution into a column, allowing the mixture to pass through the pre-packed column by gravity flow, and if the residual sample is available, loading the sample again, allowing the sample to pass through again, and collecting the flow-through solution into a centrifuge tube. The column was washed with a Binding/wash buffer of twice the column volume and the flow-through was collected until the absorbance 280nm of the flow-through was near baseline. Eluting histidine-tagged protein on the column by using an absorption Buffer with double column volume, repeating the step until the absorbance of the flow-through liquid is 280nm close to a base line, and collecting the eluent to be purified to obtain a protein solution after nickel column purification.
3. Ultrafiltration replacement
The protein solution after nickel column purification was subjected to an Amicon Ultra-15 centrifugal filtration device (Millipore Co.) and centrifuged at 10000g in batches for 3min until about 150. Mu.l of the solution remained. 300 μl PBS buffer (1×, pH 7.4) was gently added, and 10000g centrifuged to 150 μl, and repeated three times. The sample was collected from the elution ultrafiltration tube with PBS buffer (1X, pH 7.4), and the final volume was about 22ml, and 5. Mu.l of the sample was used for protein concentration measurement and SDS-PAGE protein electrophoresis detection to obtain homozygous proteins designated SP1-HCG-Fc, SP2-HCG-Fc and SP3-HCG-Fc.
As shown in FIG. 5, the purity of the purified sample was more than 90% as determined by SDS-PAGE of the three signal peptide products SP1, SP2 and SP3, and the yields of the purified protein products were 52.00mg/L,22.29mg/L and 12.07mg/L, respectively, which were substantially identical to the yields of the gray scale analysis in example 3.
The results of the above examples are combined, and a signal peptide optimization experiment is performed on the basis of the HCG-Fc fusion protein designed by the invention. The artificial engineering signal peptide SP1 (SEQ ID No. 5) is found to guide the secretory expression of eukaryotic cells of the HCG-Fc fusion protein. The artificial transformation signal peptide SP1 secretion expression quantity is obviously better than that of the antibody light chain signal peptide SP2 and the luciferase signal peptide SP3, so that the method is more suitable for large-scale industrial production and reduces the production cost.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
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